Glial reactivity is linked to synaptic dysfunction across the aging and Alzheimer’s disease spectrum

Abstract Previous studies have shown that glial and neuronal changes may trigger synaptic dysfunction in Alzheimer’s disease(AD). However, the link between glial and neuronal markers and synaptic abnormalities in the living brain is poorly understood. Here, we investigated the association between biomarkers of astrocyte and microglial reactivity and synaptic dysfunction in 478 individuals across the aging and AD spectrum from two cohorts with available CSF measures of amyloid-β(Aβ), phosphorylated tau(pTau181), astrocyte reactivity(GFAP), microglial activation(sTREM2), and synaptic biomarkers(GAP43 and neurogranin). Elevated CSF GFAP levels were linked to presynaptic and postsynaptic dysfunction, regardless of cognitive status or Aβ presence. CSF sTREM2 levels were associated with presynaptic biomarkers in cognitively unimpaired and impaired Aβ + individuals and postsynaptic biomarkers in cognitively impaired Aβ + individuals. Notably, CSF pTau181 levels mediated all associations between GFAP or sTREM2 levels and synaptic dysfunction biomarkers. These results suggest that neuronal-related synaptic biomarkers could be used in clinical trials targeting glial reactivity in AD.


INTRODUCTION
Alzheimer's disease (AD) is a neurodegenerative disorder clinically characterized by progressive cognitive and behavioral de cits 1 .The hallmark pathological features of AD are extracellular aggregates of amyloid-β (Aβ) plaques and intracellular inclusions of tau neuro brillary tangles, which are associated with brain cell dysfunction and death 2 .With the advance of in vivo biomarkers, recent studies have indicated that both glial reactivity and synaptic dysfunction constitute an integral part of the pathophysiological cascades leading to the progression of AD [3][4][5][6] .Microglia and astrocytes are brain cells that are part of the quad-partite synapse 7 , they are also involved in neuroimmune response 5,6 .Microglia are the innate immune system's primary cells in the central nervous system, which might overexpress proteins associated with AD progression, such as the triggering receptor expressed on myeloid cells 2 (TREM2) 8 .Astrocytes are cells involved in multiple physiological processes that become reactive in response to the Aβ or tau aggregation, undergoing molecular, morphological, and functional changes, including the increased expression of glial brillary acidic protein (GFAP) [9][10][11] .The importance of understanding the interplay between quad-partite synapses and classical AD proteinopathies (i.e., Aβ and tau) has been evidenced by a growing body of experimental and biomarker studies [12][13][14][15][16] .
The onset of synaptic dysfunction seems to precede neuronal loss and exhibits a stronger correlation with cognitive decline compared to Aβ plaques or tangles alone 3,4 .Reduced and compromised synaptic functionality is a key feature of individuals with cognitive impairment 17 .The process of acquiring and consolidating information in the human brain fundamentally depends on functional synapses, which are the primary orchestrators of neuronal communication 18,19 .The presynaptic biomarker growth-associated protein 43 (GAP43) appears to play a role in cognition and its levels were found to be increased in the CSF symptomatic AD patients 20,21 .Similarly, the postsynaptic marker neurogranin (Ng) is involved in facilitating long-term synaptic potentiation 22 .Elevated CSF Ng levels have been suggested as a biomarker of postsynaptic degeneration in AD 22,23 .If well validated in the context of the pathophysiological progression of AD, synaptic biomarkers can potentially be used to monitor disease progression and treatment effects 24 .It is known from the experimental literature that glial cells play fundamental roles in these processes, reducing neurotransmitters in the synaptic cleft to a su cient level, preserving physiological balance, and releasing neuroactive agents that modulate synaptogenesis 19 .Based on this, we hypothesize that phosphorylated tau, may potentially trigger in ammatory responses 25,26 and mediate interactions between glial cells and pre-and post-synaptic neuronal biomarkers.
This study aims to ll a gap in the existing literature by examining the existence of a link between glial reactivity and synaptic dysfunction biomarkers throughout the progression of AD.The insights derived from this study could be fundamental in de ning therapeutic strategies focusing on glial cell reactivity, potentially using synaptic biomarkers as secondary endpoints.
Astrocyte reactivity is associated with synaptic markers independently of Aβ.
Microglial activation is associated with synaptic markers only in the presence of Aβ.

Synaptic dysfunction as a function of glial reactivity across the AD continuum
Lowess curves corroborate previous results by demonstrating a progressive increase in pre-and postsynaptic markers as a function of astrocyte reactivity independently of Aβ (Fig. 3a), while synaptic markers increased as a function of microglial reactivity only in CU Aβ + and CI Aβ+ (Fig. 3b).When analyzing synaptic biomarkers as a function of GFAP, we observed a steeper increase in CU Aβ + cases compared to CU Aβ-and CI Aβ + .Conversely, regarding sTREM2, we nd similar increases in synaptic biomarkers in both CU Aβ + and CI Aβ + .
PTau181 mediates the association between glial reactivity and synaptic dysfunction.

DISCUSSION
This study indicates a differential association between glial responses and synaptic abnormalities in AD.Our results support that astrocyte reactivity is closely associated with pre-and post-synaptic dysfunction in patients across the aging and AD continuum.By contrast, the association between microglial activation and synaptic biomarkers was dependent on the presence of Aβ positive (Fig. 5).We also found that CSF pTau181 mediated the association between glial reactivity and synaptic dysfunction in living humans.
CSF GFAP was associated with synaptic biomarkers in CU and CI individuals independently of Aβ pathology.These results suggest early astrocyte reactivity triggering synapse damage independently of the emergence of Aβ aggregates.This observation aligns with a recent study suggesting that GFAP abnormalities precede Aβ deposition 35 .Furthermore, previous studies have shown an independent link between astrocyte reactivity and aging 36 and neurodegeneration 37 .In addition, GFAP has been associated with synaptic and neuronal degeneration in other brain conditions that are not characterized by the presence of Aβ pathology, such as Parkinson's disease 38 and traumatic brain injury 39 .It is important to highlight that although our results support that GFAP imposes synaptic dysfunction independently of Aβ, it also contributed to synaptic dysfunction in symptomatic AD individuals 40 .Together, these ndings support the notion that astrocyte reactivity is linked to a disruption of synaptic homeostasis contributing to aging and AD progression.
CSF sTREM2 was associated with synaptic biomarkers only in the presence of Aβ pathology.Our results are consistent with previous literature suggesting that Aβ pathology activates microglia in AD 41 .These studies suggest that microglial activation-related immunoresponses might potentiate Aβ aggregation 42- 45 and that Aβ pathology and microglial reactivity synergistically interact to determine cognitive deterioration in individuals across the AD spectrum 16 .Taken together, these results highlight synaptic biomarkers as potential secondary endpoints for clinical trials testing novel therapeutic strategies aimed at mitigating microglial dysfunction in patients with underlying AD pathology.
We found that CSF pTau mediates the association between glial reactivity and synaptic dysfunction.
Previous studies have shown that pTau, can potentially trigger in ammatory responses and be detected by microglia 25,26 .This process appears to mediate the link between glial reactivity and synaptic dysfunction.In this context, our ndings corroborate previous research suggesting a close connection between tau secretion, microglial activation, and synaptic damage.Speci cally, studies have shown that synaptic dysfunction precedes neurodegeneration and occurs concurrently with elevated CSF pTau in preclinical AD [43][44][45][46] .The density of neuro brillary tangles is associated with synaptic dysfunction, leading to cognitive decline 43,44 .Tau protein binds to synaptic vesicle proteins, accumulating excessively in presynaptic terminals, causing synaptic dysfunction 43 .Although evidence demonstrates independent associations between glia, tau, and synaptic damage, it is not yet understood how these pathologies interact with each other to determine pathological progression [45][46][47] .In this context, our results propose a model where tau phosphorylation mediates the deleterious effects of glial reactivity on synaptic dysfunction.These ndings suggest that reducing tau phosphorylation may potentially mitigate the effects of glial reactivity on synapses.
A strength of our study is that all results were replicated in two independent and well-characterized cohorts.It is important to acknowledge the limitations of our study.A population-based longitudinal study with multiple time points would be desirable to provide a more comprehensive understanding of the temporal dynamics and sequential relationships between biomarkers.Future mechanistic studies are needed to further corroborate our clinical ndings.Our group of participants is mainly composed of White individuals, emphasizing the importance of replicating our ndings using a more diverse sample of individuals to increase the external validity of results for the more diverse world population.
In conclusion, our ndings support a link between glial reactivity and synaptic dysfunction in living humans, which appears to be explained by pathological phosphorylation of tau.While astrocyte reactivity seems to be a partially independent phenomenon leading to synaptic dysfunction in aging and AD, the effects of microglial activation on synaptic function are determined by the emergence of Aβ pathology.
These results suggest that synaptic biomarkers hold potential as secondary endpoints for clinical trials targeting glial reactivity in aging and AD.

Participants
We included participants from two cohorts: The Translational Biomarkers in Aging and Dementia (TRIAD) cohort at McGill University, Canada (https://triad.tnlmcgill.com) and the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (https://adni.loni.usc.edu/).For TRIAD participants, the exclusion criteria were inability to speak English or French, inadequate visual and auditory capacities for neuropsychologic assessment, active substance abuse, major surgery, recent head trauma, medical contraindication for positron emission tomography (PET) or magnetic resonance imaging (MRI), and neurological, psychiatric, or systemic comorbidities that were not adequately treated with a stable medication regimen 13 .ADNI inclusion and exclusion criteria are available at https://adni.loni.usc.edu/.
Recruited participants were between the ages of 55 and 90 years, completed at least six years of education, were uent in Spanish or English, had a Hachinski ischemic score less than or equal to four, and had screening/baseline MRI scans without evidence of infection, infarction, or other focal lesions (individuals with multiple lacunes or lacunes in a critical memory structure were excluded).We assessed a total of 480 individuals: 107 from the TRIAD cohort [64 cognitively unimpaired (CU), 43 cognitively impaired (CI)], and 373 from the ADNI cohort [113 CU, and 260 CI].We included all participants from these cohorts who were 50 years or older, who had available CSF GFAP, soluble TREM2 (sTREM2), GAP43, Ng, Aβ42, Aβ42/40, and tau protein phosphorylated at threonine 181 (pTau181) values.CU and CI were de ned as described elsewhere 27 : CU individuals had no memory complaints and a Mini-Mental State Examination (MMSE) score between 24 and 30, while CI individuals (including patients with mild cognitive impairment (MCI) and dementia) had an MMSE score below 24.We de ned Aβ positivity using a published validated cutoff for CSF Aβ42 abnormality for each cohort 28,29 .The studies were approved by institutional review boards of all participating centers, and written informed consent was obtained from all participants or their authorized representatives were applicable.

CSF measurement
In the TRIAD cohort, CSF data was analyzed at the University of Gothenburg, Sweden blinded to participant clinical information.CSF GFAP levels were quanti ed on the Simoa HD-X (Quanterix) using the commercial single-plex assay (No. 102336) 30 ; CSF sTREM2 concentration was measured using a Meso-Scale Discovery assay, as described previously 30 ; CSF pTau181 and Aβ42/40 were quanti ed by LUMIPULSE G1200 (Fujirebio) 28 ; synaptic biomarkers, GAP43 and Ng were measured using in-house immunoassays developed at the University of Gothenburg 31 .The ADNI cohort procedure manual describes the CSF collection process (http://www.adni-info.org/).CSF sTREM2 measurements were performed using ELISA by the laboratory of C. Haass at the DZNE Munich as described previously 32 ; CSF Aβ42 and CSF pTau181 were quanti ed using the Elecsys assays (Roche Diagnostics) 29 ; CSF GAP-43 quanti cation was performed using an in-house ELISA method at the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital (Mölndal, Sweden) 33 ; CSF Ng concentration was measured by electrochemiluminescence as described previously 21,32 .

Statistical analysis
The values were standardized using z-scores centered on the mean within each cohort.Continuous variables were evaluated for differences between groups using analysis of variance (ANOVA) with post hoc Tukey comparison.For categorical or ordinal variables, the Kruskal-Wallis test was used followed by the Mann-Whitney U post hoc test.We examined the associations between variables using linear regression models adjusted for sex and age.We used the locally weighted scatterplot smoothing (Lowess) method involving 2,000 iterations with a smoothing span of 1.00 to model biomarker trajectories 13 .The mediation analysis was conducted for the observation of both direct and indirect effects.Here, the direct effect refers to the relationship between glial markers and synaptic markers, while the indirect effect, mediated by pTau181, captured the association between these variables.The signi cance of the mediation was assessed by calculating bias-corrected 95% con dence intervals using bootstrapping with 500 resamples 34 .Biomarker values were standardized using mean-centered z-scores within each cohort, adjusted for sex and age, allowing integration of the TRIAD and ADNI data for mediation analyses, in addition, the model was adjusted with the cohort factor.Biomarker values were standardized using z-scores centered on the mean within each cohort enabling the integration of TRIAD and ADNI cohorts for mediation analyses.P < 0.05 indicated a signi cant difference.Statistical analyses were conducted using the R software (version 4.0.2,http://www.r-project.org/).

Con ict of Interest
Dr Therriault has served as a paid consultant for the Neurotorium educational platform and for Alzheon.
Dr Ashton received payment for lectures from Biogen, BioArtic, Eli-Lily, and Quanterix.Dr. Karikari reported receiving grants from the National Institutes of Health and receiving personal fees from the University of Wisconsin-Madison and the University of Pennsylvania outside the submitted work; in addition, Dr Karikari had a patent for WO2020193500A1 issued.

Figures Figure 1 CSF
Figures

Figure 5 Schematic
Figure 5 Dr Zetterberg reported receiving personal fees from Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, LabCorp, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave; receiving personal fees for sponsored lectures from Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche outside the submitted work; and being a cofounder of Brain Biomarker Solutions, which is a part of the GU Ventures Incubator Program outside submitted work.Dr Blennow reported having served as a consultant and at advisory boards for Acumen, ALZPath, BioArctic, Biogen, Eisai, Eli Lilly and Co, Moleac Pte Ltd, Novartis, Ono Pharma, Prothena, Roche Diagnostics, and Siemens Healthineers; having served on data monitoring committees for Julius Clinical and Novartis; having given lectures, produced educational materials, and participated in educational programs for AC Immune, Biogen, Celdara Medical, Eisai, and Roche Diagnostics; and being a cofounder of Brain Biomarker Solutions, which is a part of the GU Ventures Incubator Program, outside the submitted work.Dr Zimmer served in the SAB of Novo Nordisk, serves on SAB of Next Innovative Therapeutics (Nintx) and serves on the SAB and is a Co-founder of MASIMA.Dr Rosa-Neto served in the SAB of Novo Nordisk, Eisai, and Ely Lilly and as a Consultant in Eisai and Cerveau radiopharmaceuticals.No other disclosures were reported.